Cytochrome P-450scc catalyses the conversion of cholesterol to pregnenolone by the sequential hydroxylation of the side chain of cholesterol. This occurs at a single active site and produces 22R-hydroxycholesterol and 22R-20alpha-dihydroxycholesterol as intermediates. To further define the active site of human and bovine cytochromes P-450scc, we have examined the kinetics of the conversion of structural analogues of cholesterol with modified side chains, to pregnenolone. Analysis of the side-chain cleavage of analogues of cholesterol modified at C22 confirmed the high degree of structural specificity for the 22R position by cytochrome P-450scc, the major effect being on the turnover number (k(cat)) rather than on binding. The analogues of cholesterol that had a polar group at C24, C25 or C26 had much lower K(m) values and generally lower k(cat) values than the non-polar analogues which were tested. K(m) values of the polar analogues were 3-25-times lower than the K(m) for cholesterol and k(cat) values were also much lower than the k(cat) values for cholesterol, particularly for the human enzyme. The data suggest that the tight binding of the analogues with a hydroxyl or ketone group at C24, C25 or C26 places C20 and C22 in a poor orientation relative to the heme group for hydroxylation to occur. Many of the polar analogues which were tested are postulated regulators of cellular cholesterol metabolism. Several of these analogues are good substrates for bovine and human cytochromes P-450scc at low substrate concentration, as determined from their k(cat)/K(m) values. This study also indicates that the active site of cytochrome P-450scc is well conserved between bovine and human cytochromes. However, small species differences are evident since lower k(cat), values relative to the k(cat) of cholesterol are observed for some polar side-chain analogues of cholesterol with the human enzyme.
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1993|